High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.


Measurement of oxidation status of HMGB1 by biotin switch assay
A biotin labeling assay was used to investigate the oxidation state of the cysteine residues on recombinant HMGB1 (R&D Systems) as previously described (1).Briefly, recombinant HMGB1 was either reduced by sodium dithionite (100 mM, 15 min), oxidized by hydrogen peroxide (H2O2, 100 μM, 15 min), or left untreated.
The exposed free thiol groups in each of these samples were labeled with EZ-Link Maleimide-PEG2-Biotin, 100μM (Thermo Scientific) by incubating for 2 hours at 40°C.Samples were loaded onto 10% SDS page gel and electrophoresis was performed as described(98).Biotin was detected by streptavidin binding (1:5000, Li-Cor) images were captured by Odyssey CLx Imager (Li-Cor).Semiquantitative analysis was performed using Image Studio Lite software.Biotin quantification was normalized to quantification of HMGB1 protein within each lane (using monoclonal anti-HMGB1 antibody 1:500 dilution, Biolegend).This approach provided insight into the relative oxidation level of recombinant HMGB1 used throughout the manuscript and allowed for semiquantitative analysis of the biotin-labeled thiol groups.

Quantification of total P2Y12 in human platelets by Western Blot
Isolated human platelets (4×10 7 cells/mL) were treated with either HMGB1 (10 µg/ml) or vehicle for 20 minutes at room temperature and were lysed by repeated freeze-thaw.SDS-PAGE gel electrophoresis was performed as described(98).After transfer to nitrocellulose, membranes were blocked with 5% milk in TBST.P2Y12 was probed with primary rabbit anti-P2Y12 mAb (1:1000 dilution) in 2.5-5% milk and incubated at 4°C overnight; rabbit anti-vinculin mAb (1:500 dilution; ThermoFisher Scientific #700062) was probed as loading control.Membranes were washed and incubated with horseradish peroxidase conjugated secondary antibody followed by ECL reagent as reported(99).
Images were captured by chemiluminescence on a Kodak X-OMAT 2000 Processor.
Semiquantitative analysis was performed using Image Lite software.

Supplemental
Figure S1 Supplemental Figure S1: Evaluating the impact of the redox status of recombinant HMGB1 on platelet activation.(A) Biotin signal normalized to HMGB1 protein for untreated recombinant HMGB1 (R&D Systems) and for recombinant HMGB1 that was reduced and oxidized, respectively, and (B) corresponding quantifications.Higher biotin signal denotes more free thiols (SH) as seen with the reduced form.Lower and intermediate biotin signals are expected for the oxidized and disulfide forms, respectively, where the sulfonate groups (HO3S) and disulfide S-S bond limits available binding sites.n=5 and are analyzed by one-way ANOVA.(C) Platelet activation with stimulation of each redox form of HMGB1 (10 μg/ml).n=3 and are analyzed by one-way ANOVA.All data are mean ± SEM * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001.

Table 1 : Key Antibodies and Reagents 94 Legend forTable S1 :
A comprehensive list of key reagents and antibodies used within this study are listed here.